Calcium is an important signaling molecule for many normal physiological processes in the human body. These include electrical signaling in the nervous system, as well as controlling heart and smooth muscle contraction, and hormone release. The entry of calcium into cells is regulated by a diverse set of proteins called calcium channels.
A fundamental role of Ca2+ channels is to translate an electrical signal on the surface membrane into a chemical signal within the cytoplasm, which, in turn, activates many important intracellular processes including contraction, secretion, neurotransmission and regulation of enzymatic activities and gene expression. Tsien et al., (1988), Trends Neurosci., vol. 11, pp. 431-438. Continuing studies have revealed that there are multiple types of Ca2+ currents as defined by physiological and pharmacological criteria. See, e.g., Catterall, W. A., (2000) Annul Rev. Cell Dev. Biol., 16:521-55; Llinas et al, (1992) Trends Neurosci, 15; 351-55; Hess, P. (1990) Ann. Rev. Neurosci. 56:337; Bean, B. P. (1989) Ann. Rev. Physiol. 51:367-384; and Tsien et al. (1988) Trends Neurosci. 11:431-38. In addition to exhibiting distinct kinetic properties, different Ca2+ channel types can be localized on different regions of a cell and have complex morphology. The calcium in nerve cells plays an important role in delivering signals between nerve cells. Voltage activated calcium channels play important roles including neuroexcitation, neurotransmission and hormone secretion, and regulation of gene transcription through Ca-dependent transcription factors.
Voltage dependent calcium channels have been classified by their electrophysiological and pharmacological properties (McCleskey, E. W. et al. Curr Topics Membr (1991) 39:295-326, and Dunlap, K. et al. Trends Neurosci (1995) 18:89-98). Voltage-gated calcium channels can be divided into Low Voltage Activated calcium channels (LVA), that are activated at a lower voltage, and High Voltage Activated (HVA) calcium channels, that are activated at a higher voltage with respect to typical resting membrane potentials. HVA channels are currently known to comprise at least three groups of channels, known as L-, N- and P/Q-type channels. These channels have been distinguished from one another electrophysiologically as well as biochemically on the basis of their pharmacology and ligand binding properties. The L-, N-, P/Q-type channels activate at more positive potentials (high voltage activated) and display diverse kinetics and voltage-dependent properties. To date, only one class of low-threshold calcium channels is known, the T-type calcium channels. These channels are so called because they carry a transient current with a low voltage of activation and rapid inactivation. (Ertel and Ertel (1997) Trends Pharmacol. Sci. 18:37-42.). In general, T-type calcium channels are involved in the generation of low threshold spikes to produce burst firing (Huguenard, J. R., Annul Rev. Physiol., 329-348, 1996).
Three genes are known to encode pore forming subunits of T-type calcium channels; CACNA1G (alpha1G, Cav3.1), CACNA1H (alpha1H, Cav3.2), and CACNA1I (alpha1I, Cav3.3) (see Perez-Reyes, Physiol Rev. 2003 83:117-61).
T-type calcium channels are located in the nervous system, cardiac & vascular smooth muscle; as well as a variety of endocrine cell types (see Perez-Reyes, Physiol Rev. 2003 83:117-61). Generally, T-type channels are believed to be involved in electrical pacemaker activity, low-threshold calcium spikes, neuronal oscillations and resonance (Perez-Reyes, Physiol Rev. 2003 83:117-61). The functional roles for T-type calcium channels in neurons include, membrane depolarization, calcium entry and burst firing. (White et al. (1989) Proc. Natl. Acad. Sci. USA 86:6802-6806). Functionally unique calcium channels allow for temporal and spatial control of intracellular calcium and support regulation of cellular activity.
T-type calcium channels have more negative activation ranges and inactivate more rapidly than other calcium channels. When the range of membrane potentials for activation and inactivation overlap, T-type calcium channels can undergo rapid cycling between open, inactivated, and closed states, giving rise to continuous calcium influx in a range of negative membrane potentials where HVA channels are not normally activated. The membrane depolarizing influence of T-type calcium channel activation can become regenerative and produce calcium action potentials and oscillations.
In addition to the variety of normal physiological functions mediated by calcium channels, they are also implicated in a number of human disorders. For example, changes to calcium influx into neuronal cells may be implicated in conditions such as epilepsy, stroke, brain trauma, Alzheimer's disease, multiinfarct dementia, other classes of dementia, Korsakoff's disease, neuropathy caused by a viral infection of the brain or spinal cord (e.g., human immunodeficiency viruses, etc.), amyotrophic lateral sclerosis, convulsions, seizures, Huntington's disease, amnesia, pain transmission, cardiac pacemaker activity or damage to the nervous system resulting from reduced oxygen supply, poison or other toxic substances (Goldin et al., U.S. Pat. No. 5,312,928). Other pathological conditions associated with elevated intracellular free calcium levels include muscular dystrophy and hypertension (Steinhardt et al., U.S. Pat. No. 5,559,004).
Low threshold spikes and rebound burst firing characteristic of T-type calcium currents is prominent in neurons from inferior olive, thalamus, hippocampus, lateral habenular cells, dorsal horn neurons, sensory neurons (DRG, nodose), cholinergic forebrain neurons, hippocampal intraneurons, CA1, CA3 dentate gyros pyramidal cells, basal forebrain neurons, amygdala neurons (Talley et al., J. Neurosci., 19: 1895-1911, 1999) and neurons in the thalamus (Suzaki and Rogawski, Proc. Natl. Acad. Sci. USA 86:7228-7232, 1998). As well, T-type channels are prominent in the some and dendrites of neurons that reveal robust Ca dependent burst firing behaviors such as the thalamic relay neurons and cerebellar Purkinje cells (Huguenard, J. R., Annul Rev. Physiol., 329-348, 1996). Consequently, improper functioning of these T-type calcium channels has been implicated in arrhythmias, chronic peripheral pain, inappropriate pain transmission in the central nervous system.
The reduction of in vivo hyperalgesic responses to thermal or mechanical stimuli induced by chemical agents (i.e. reducing agents, capsaicin) or experimental nerve injury (i.e. chronic constriction injury; spinal nerve ligation) by known T-type calcium channel antagonists mibefradil and/or ethosuximide suggests a role of the T-type calcium channels in peripheral nerve pain signaling (Todorovic, Neuron, 2001, 31:75-85; Todorovic and Lingle, J. Neurophysiol. 79:240-252, 1998, Flatters S J, Bennett G J. Pain. 2004 109:150-61; Dogrul et al; Pain. 2003 105:159-68; Matthews and Dickenson. Eur J Pharmacol. 2001 415:141-9). Furthermore, intrathecal administration of antisense oligonucleotides to alphalH (Cav3.2) T-type calcium channels in rodents has recently been shown to selectively inhibit the functional expression of T-type calcium currents in sensory neurons and reverse hyperalgesic, and allodynic, responses induced by experimental nerve injury (Bourinet et al EMBO J. 2005 24:315-24). Gene knockout of alpha1G (Cav3.1) T-type channels in mouse CNS is reported to increase the perception of visceral pain (Kim et al. Science. 2003 302:117-9).
T-type calcium channels promote oscillatory behavior, which has important consequences for epilepsy. The ability of a cell to fire low threshold spikes is critical in the genesis of oscillatory behavior and increased burst firing (groups of action potentials separated by about 50-100 ms). T-type calcium channels are believed to play a vital role in absence epilepsy, a type of generalized non-convulsive seizure. The evidence that voltage-gated calcium currents contribute to the epileptogenic discharge, including seizure maintenance and propagation includes: 1) a specific enhancement of T-type currents in the reticular thalamic (nRT) neurons which are hypothesized to be involved in the genesis of epileptic seizures in a rat genetic model for absence epilepsy (Tsakiridou et al., J. Neurosci., 15: 3110-3117, 1995); 2) antiepileptics against absence petit mal epilepsy (ethosuximide and dimethadione) have been shown at physiologically relevant doses to partially depress T-type currents in thalamic neurons (Courter et al., Ann. Neurol., 25:582-93, 1989; U.S. Pat. No. 6,358,706 and references cited therein), and; 3) T-type calcium channels underlie the intrinsic bursting properties of particular neurons that are hypothesized to be involved in epilepsy (nRT, thalarnic relay and hippocampal pyramidal cells) (Huguenard).
The T-type calcium channels have been implicated in thalamic oscillations and cortical synchrony, and their involvement has been directly implicated in the generation of cortical spike waves that are thought to underlie absence epilepsy and the onset of sleep (McCormick and Bal, Annul Rev. Neurosci., 20: 185-215, 1997). Oscillations of neural networks are critical in normal brain function such during sleep-wave cycles. It is widely recognized that the thalamus is intimately involved in cortical rhythmogenesis. Thalamic neurons most frequently exhibit tonic firing (regularly spaced spontaneous firing) in awake animals, whereas phasic burst firing is typical of slow-wave sleep and may account for the accompanying spindling in the cortical EEG. The shift to burst firing occurs as a result of activation of a low threshold Ca2+ spike which is stimulated by synaptically mediated inhibition (i.e., activated upon hyperpolarization of the RP). The reciprocal connections between pyramidal neurons in deeper layers of the neocortex, cortical relay neurons in the thalamus, and their respective inhibitory interneurons are believed to form the elementary pacemaking circuit.
Tremor can be controlled through the basal ganglia and the thalamus, regions in which T-type calcium channels are strongly expressed (Talley et al J Neurosci. 1999 19:1895-911). T-type calcium channels have been implicated in the pathophysiology of tremor since the anti-epileptic drug ethosuximide is used for treating tremor, in particular, tremor associated with Parkinson's disease, essential tremor, or cerebellar disease (U.S. Pat. No. 4,981,867; D. A. Prince).
It is well documented that cortisol is the precursor for glucocorticoids and prolonged exposure to glucocorticoids causes breakdown of peripheral tissue protein, increased glucose production by the liver and mobilization of lipid from the fat depots. Furthermore, individuals suffering from anxiety and stress produce abnormally high levels of glucocorticoids. Consequently, drugs that would regulate these levels would aid in the treatment of stress disorders. In this regard, the observations (Enyeart et al., Mol. Endocrinol., 7:1031-1040, 1993) that T-type channels in adrenal zone fasciculata cells of the adrenal cortex modulate cortisol secretion will greatly aid in the identification of such a therapeutic candidate.
T-type calcium channels may also be involved sperm production. Sertoli cells secrete a number of proteins including transport proteins, hormones and growth factors, enzymes which regulate germinal cell development and other biological processes related to reproduction (Griswold, Int. Rev. Cytol., 133-156, 1988). While the role of T-type calcium channels remains to be fully elucidated, it is believed that they may be important in the release of nutrients, inhibin B, and/or plasminogen activator and thus may impact sperm production. According to researchers, the inhibition of T-type calcium channels in sperm during gamete interaction inhibits zona pellucida-dependent Ca2+ elevations and inhibits acrosome reactions, thus directly linking sperm T-type calcium channels to fertilization.
In view of the above, pharmacological modulation of T-type calcium channel function is very important and therapeutic moieties capable of modulating T-type currents may find utility in the practice of medicine, i.e., calcium channel blockers for the treatment of pain, epilepsy, hypertension, and angina pectoris etc. Compounds identified thereby may be candidates for use in the treatment of disorders and conditions associated with T-channel activity in humans and animals. Such activities include, but are not limited to, those involving a role in muscle excitability, secretion and pacemaker activity, Ca2+ dependent burst firing, neuronal oscillations, and potentiation of synaptic signals, for improving arterial compliance in systolic hypertension, or improving vascular tone, such as by decreasing vascular welling, in peripheral circulatory disease, and others. Other disorders include, but are not limited to hypertension; cardiovascular disorders (e.g. myocardial infarct, cardiac arrhythmia, heart failure and angina pectoris); neurological disorders (e.g. epilepsy, pain, schizophrenia, depression and sleep); peripheral muscle disorders; respiratory disorders; and endocrine disorders. The present invention meets these and other needs in the art.